Development of Genetic Knock-out Systems for Clostridia
John T. Heap, Stephen T. Cartman, Oliver J. Pennington, Clare M. Cooksley, Jamie C. Scott, Ben Blount, David Burns and Nigel P. Minton
from: Clostridia: Molecular Biology in the Post-genomic Era (Edited by: Holger Brüggemann and Gerhard Gottschalk). Caister Academic Press, U.K. (2009)
Despite the medical and industrial importance of the genus Clostridium our understanding of their basic biology lags behind that of their more illustrious counterpart, Bacillus. The advent of the genomics era has provided new insights, but full exploitation of the data becoming available is being hindered by a lack of mutational tools for functional genomic studies. Thus, in the preceding decades the number of clostridial mutants generated has been disappointingly low. On the one hand, the absence of effective transposon elements has stymied random mutant generation. On the other hand, the construction of directed mutants using classical methods of recombination-based, allelic exchange has met with only limited success. Indeed, in the majority of clostridial species mutants are largely based on integration of plasmids by a Campbell-like mechanism. Such single crossover mutants are unstable. As an alternative, recombination-independent strategies have been developed that are reliant on retargeted group II intron. One element in particular, the ClosTron, has been devised which provides the facility for the positive selection of mutants. ClosTron-mediate mutant generation is extremely rapid, highly efficient and reproducible. Moreover the mutants made are extremely stable. Its deployment considerably expands current options for functional genomic studies in clostridia read more ...